We propose study of the mechanism by which "high voltage" pulses (about 20 to 800 V across the skin) cause greatly enhanced transport of charged molecules across the main barrier of the human skin, the stratum corneum. AIMS. Our goals are directed towards testing the hypothesis that aqueous pathways are created in the stratum corneum, and involve experiments which use a series of charged and uncharged fluorescent molecules to measure fluxes and stored amounts of these molecules under in vitro conditions. SIGNIFICANCE. The lipid-rich stratum corneum (SC; outermost, dead layer of the human skin) is comprised of dead corneocytes surrounded by multiple, parallel bilayer membranes, and protects the body from dehydration, entry of toxic molecules and infection. However the SC also presents a barrier for transdermal drug delivery and analyte extraction. High voltage pulsing is expected to create new aqueous pathways across the stratum corneum, thereby increasing both the amount of molecular transport and degree of control for transdermal drug delivery. Of particular interest is the transport of charged molecules (e.g., peptides) which presently cannot be delivered in large amounts. The opposite, complimentary process of extracting analytes (e.g. glucose) from the subcutaneous interstitial fluid would be of great value for "non-invasive clinical chemistry" assays. PREVIOUS WORK. We have shown that significant fluxes of charged molecules can cross the stratum corneum if a series of short "high voltage" pulses are applied. Limited animal studies and prior medical use of "high voltage" pulses together suggest that insignificant damage occurs for some pulsing conditions, but that for larger and/or longer pulses there is tissue damage. Other work with cell suspensions, and with theoretical models for electroporative transport, suggests that electrophoresis through transient aqueous pores in cellular bilayer membranes, and in the multilamellar bilayers of the stratum corneum, may be the dominant mechanism for the transport of charged molecules through electroporated bilayer membranes. METHODS. Under in vitro conditions, we will carry out three simultaneous measurements on each skin preparation: [1] the flux of a first fluorescent molecule, [2] the flux of a second fluorescent molecule, and [3] a passive electrical property (e.g. skin impedance or resistance) measurement. This approach addresses the important issue of skin preparation variability, while providing critical quantitative information by using fluorescent molecules with different size, charge and water/lipid solubility. These experiments will be designed to test versions of the general hypothesis that aqueous pathways are created by "high voltage" pulsing, and that electrophoresis (and possibly electro-osmosis and diffusion) through these pathways can account for the tremendous flux increases. A companion theoretical modeling effort will provide explicit predictions that allow direct testing of the "aqueous pathway creation" hypothesis.